Glucosyl Platinum(II) Complexes Inhibit Aggregation of the C-Terminal Region of the Aβ Peptide

Neurodegenerative diseases are often caused by uncontrolled amyloid aggregation. Hence, many drug discovery processes are oriented to evaluate new compounds that are able to modulate self-recognition mechanisms. Herein, two related glycoconjugate pentacoordinate Pt(II) complexes were analyzed in their capacity to affect the self-aggregation processes of two amyloidogenic fragments, Aβ21–40 and Aβ25–35, of the C-terminal region of the β-amyloid (Aβ) peptide, the major component of Alzheimerʼs disease (AD) neuronal plaques. The most water-soluble complex, 1Ptdep, is able to bind both fragments and to deeply influence the morphology of peptide aggregates. Thioflavin T (ThT) binding assays, electrospray ionization mass spectrometry (ESI-MS), and ultraviolet–visible (UV–vis) absorption spectroscopy indicated that 1Ptdep shows different kinetics and mechanisms of inhibition toward the two sequences and demonstrated that the peptide aggregation inhibition is associated with a direct coordinative bond of the compound metal center to the peptides. These data support the in vitro ability of pentacoordinate Pt(II) complexes to inhibit the formation of amyloid aggregates and pave the way for the application of this class of compounds as potential neurotherapeutics.


■ INTRODUCTION
In Alzheimerʼs disease (AD) pathophysiology, the β-amyloid (Aβ) peptide represents the prevalent component of senile plaques 1 even if it is present at the early phases of life, and in its monomeric form, it can act as a positive regulator of the presynaptic release in hippocampal neurons. 2 Aβ is a polypeptide spanning 1−40 or 42 residues that is mostly intrinsically disordered. 3 Through a self-assembly process that is typical of amyloid aggregation, it forms different Aβ oligomers, endowed with diverse levels of order that represent key pathogenic species in AD. 4 Amyloid assemblies induce synaptic dysfunction and neuronal death 5 and oxidative damage and inflammation, which further corroborate the progression of the disease. 6 The presence of metals like Zn, Fe, Cu, and Al inside amyloid plaques enhances Aβ-induced oxidative damage and its aggregation level; thus, a chelation approach to directly target metals in the brain can be conceived as a way to reduce harmful consequences of metal/fibril accumulation. 7 However, the most powerful therapeutic approach for AD is based on the inhibition of the aggregation of the Aβ peptide 8 and great efforts have been devoted to the identification of molecules capable of inhibiting its self-recognition. It has been shown that these compounds can have different origins (synthetic, natural) and chemical nature (phenols, peptide, antibodies, small molecules, etc.). 9−12 Even though many trials are actually ongoing, especially on natural compounds, 13 no drugs have entered into clinical use yet: this can be mainly due to the inability of targeting protein interfaces without regular secondary structures that could be assumed as templates to design inhibitors. 14,15 Recently, multivalent systems (as dendrimers) were investigated to gain access to different protein subregions. 16 Indeed, different Aβ regions contribute to amyloid aggregation: the Nterminus, 17 hydrophobic core, 18 so-called hinge and turn regions, 19 and C-terminus. 20, 21 Experimental data indicated that the C-terminal region of Aβ can be addressed by the cyclohexanehexol scaffold: indeed, the scyllo-inositol compound interferes with the fibrillization process and competes with endogenous phosphatidylinositol for binding to the Aβ polypeptide, appearing as a promising therapeutic agent, currently in Phase II trials. 22 The unique properties exhibited by transition-metal complexes as drugs, including their tunability in the oxidation and the coordination states, allow them to enter into many pharmaceutical applications. 23 −25 In the amyloid context, they can be considered as good starting compounds for the development of novel neuroprotective agents. 26,27 The stability and inertness of Pt(II) and Ru(III) 28 complexes allowed a wide range of investigations with different amyloid systems: phenanthroline (phen)−Pt(II) complexes with two monodentate ligands (e.g., chlorides) inhibit the aggregation of Aβ 1−40 and its N-terminal region 29−31 as well as of prion protein (PrP) fragments. 32,33 Polyoxometalate derivatives of Pt 34 and V 35 suppress amyloid aggregation in a mechanism involving multiple interactions: (i) the coordination of the metal, (ii) electrostatic, (iii) hydrogen, and (iv) van der Waals forces. Octahedral Co compounds, 36 as well as square-planar Pt complexes bearing polyaromatic ligands, interact with the Aβ peptide via π−π stacking. 37 Furthermore, hetero-multinuclear Pt−Ru complexes are able to revert amyloidosis: they regulate amyloid-induced cytotoxicity in insulinoma β-cells and significantly increase cell viability. 38 Traditionally, due to the presence of His residues in its sequence, the N-terminal region of the Aβ peptide was considered the main target for inhibition studies of Aβ aggregation by metal compounds. 39 Conversely, with the aim to deepen the druggability of C-terminus by transition-metal complexes, in our recent investigations we assumed as amyloid model the fragment spanning residues 21−40 of Aβ (Aβ 21−40 , Table 1), 40−42 which is also deeply involved in the mechanism of aggregation of the entire Aβ peptide. Herein, we also studied the behavior of a shorter Aβ fragment spanning residues 25−35 (Aβ 25−35 , Table 1). It has been shown that this peptide is the most cytotoxic among the known Aβ peptides. 43 Very recently, we have investigated the ability of a series of square-planar Pt(II) complexes to inhibit the aggregation of amyloid peptides. 40−42 Among the investigated systems, the pyridine-based platinum(II) complex, called Pt-terpy, exhibited good inhibitory effects of amyloid aggregation, also allowing, for its better water solubility when compared to other investigated complexes, to get insights into the mechanism of action of these types of molecules: the presence of the Pt complex stabilized soluble β-structures of the Aβ 21−40 peptide. 44 Some of us also designed new pentacoordinate glycoconjugate platinum(II) complexes that can act as anticancer compounds. 45 Sugar ligands were introduced aiming to enhance their biocompatibility, aqueous solubility, and recognition by cancer cells through the "Warburg effect". 46,47 The coordinative saturation is also an important stereoelectronic requisite that improves their general stability. In particular, the peracetylated NHC complex 1Pt in Figure 1, prepared along with its deprotected counterpart 1Pt dep , showed high activity and selectivity toward a panel of cell lines. 48 Here, we present investigations focused on the ability of the two complexes to act as effective inhibitors of aggregation of Aβ peptides reported in Table 1. The ability of 1Pt dep to inhibit the aggregation of Aβ peptides was confirmed via a range of spectroscopic and biophysical techniques. Initially, ThT was employed as a typical amyloid dye, which is able to bind to amyloid prefibrils, inducing a strong fluorescence signal at ∼482 nm when excited at 440 nm. 49,50 The overlays of ThT fluorescence emission profiles of the investigated peptides in the absence and presence of 1Pt and 1Pt dep over time are reported in Figure 2.
Aβ peptide aggregation was investigated in the presence of metal complexes at peptide-to-metal molar ratios of 1:1 and 1:5. A reduction of aggregation in the presence of metal compounds is observed for almost all samples; fluorescence quenching is more evident in the case of Aβ 25−35 . Comparing the effects of Pt complexes on the Aβ 21−40 aggregation, 1Pt dep exhibits greater suppressive effects ( Figure 2A) with respect to 1Pt ( Figure 2C). 1Pt dep decreases aggregation of 60 and 90% at 1:1 and 1:5 peptide-to-metal molar ratios, respectively. Under similar experimental conditions (with the addition of dimethyl sulfoxide (DMSO), 2% (v/v), required for dissolution of the compound), the reduction of aggregation is less evident when the peptide is treated with 1Pt: it is about 35 and 20% at 1:1 and 1:5 peptide-to-metal compound molar ratios, respectively ( Figure 2C). The differences between the behavior of the two Pt complexes are more evident when the ThT profiles of Aβ 25−35 are compared: when the peptide is incubated with 1Pt dep , the reduction of the aggregated fraction (70%) is essentially independent of the equivalents of the complex that were used ( Figure 2B); on the contrary, when the peptide is treated with 1Pt, the behavior is similar to that observed in the experiments carried out with Aβ 21−40. The suppressive effect of Aβ 25−35 aggregation exerted by 1Pt is less significant than that of 1Pt dep : the level of aggregation inhibition is comparable to that exhibited by 1Pt dep only Table 1. Sequences of the Aβ 1-42 Peptide and Peptides Investigated in this Study and Derived from its C-Terminal Domain Inorganic Chemistry pubs.acs.org/IC Article when the peptide-to-metal compound molar ratio is 1:5 ( Figure 2D). Noticeably, a preliminary experiment employing the entire Aβ 1−42 sequence as aggregating polypeptide, 51 reported in Figure S1, confirmed the ability of 1Pt dep to inhibit amyloid aggregation, at a 1:5 Aβ 1−42 /1Pt dep molar ratio. Future experiments will detail the different involvements of Nand C-terminal regions in this inhibitory process.  Tables 2 and 3, respectively. 1Pt dep appears to be able to bind to both peptides by substituting the equatorial 2,9-dimethyl-1,10-phenanthroline (Dmphen) and ethylene ligands, as demonstrated by the presence of the peak at m/z 1190.06 (Table 2, Table 2) and 2411.73 Da (component C in Table 3) and corresponding to the adducts generated by Aβ 21−40 and Aβ 25−35 . In addition to these common features, several relevant differences emerge from the analysis of the spectra: the first difference is regarding the kinetics of adduct formation. Indeed, contrary to that found for Aβ 21 Figure 3B, Table 2).
The analysis of the b series in the spectra of the peptide with 1Pt dep provided insights into the peptide fragments mainly involved in the adduct formation. In the spectra of Aβ 25−35 with 1Pt dep (Figure 4 44 ESI-MS analysis was also carried out by incubating βpeptides with the 1Pt complex. In this case, no peaks deriving from adducts were detected over the time for Aβ 21−40 ( Figure  S3). A peak was found only for Aβ 25−35 , in the spectral background, consistent with a 2:1 stoichiometry and compatible with an adduct of the Pt(II) complex with both the axial ligands ( Figure S4). These findings confirm the results already described by Annunziata et al.: 48 small variations in terms of the ligand structure, such as the presence of protecting groups, are able to substantially modify the binding capacity of a metal compound toward the same biomolecule. On the basis of these results, we further investigated only the ability of 1Pt dep to modulate the amyloid aggregation of Aβ peptides.
Spectroscopic Investigations of Adducts with 1Pt dep . Ultraviolet−visible (UV−vis) absorption spectroscopy was employed to detect potential variations of the ligand field of 1Pt dep induced by the presence of amyloid peptides. In agreement with literature studies on Pt(II)−diimine complexes, 52,53 the spectra are characterized by the presence of the π → π* intraligand and Pt(5d) → π* metal-to-ligand charge transfer (MLCT) transitions in the 200−400 nm region. As reported in Figure 5 upper panel, an enhancement of absorbances upon increasing the amounts of both Aβ 25−35 ( Figure 5A) and Aβ 21−40 ( Figure 5B) is observable: this suggests that a mechanism of substitution of ligands around the Pt center occurred. This titration, in the case of Aβ 25−35 , allowed us to estimate EC 50 = 93.7 ± 0.2 μM, through the fitting of absorbance values at 330 nm (inset of Figure 5A). This value is comparable to that observed in other studies of metal complex/amyloid peptide systems. 41,44 Data fitting did  Figure 5B). To evaluate if the presence of 1Pt dep could have effects on the conformation of Aβ peptides, we registered CD spectra of freshly prepared samples and of the peptides incubated for 24 h with the metal compound. Spectra are reported in Figure 5. At t = 0, Aβ 21−40 presented a substantial random coil profile, while Aβ 25−35 presented a mixture of random coil and β-sheet signals; 54 after 24 h, for both sequences, a clear conformational transition toward β-structures occurred ( Figure 5C,E), as often observed during amyloid aggregation. 44,55 Spectra registered in the presence of 1Pt dep , which exhibits a nonnull Cotton effect ( Figure S5), at two different times, indicate the absence of the minimum at 220 nm typical of β-sheet structures ( Figure  5D,F). This finding suggests that the presence of the Pt  The m/z experimental values, experimental and theoretical monoisotopic molecular weights (MWs), and relative species are reported. In the "component" column, the labels of species in Figure 3 are reported. Me: methyl ligand; sugar: glucosyl ligand.  The m/z experimental values, experimental and theoretical monoisotopic molecular weights, and the relative species are reported. In the component column, the labels of species in Figure 4 are reported. Me: methyl ligand; sugar: glucosyl ligand.  We first run one-dimensional (1D) [ 1 H] spectra, reported in Figure 6, of Aβ 25−35 alone at t = 0 and 4 h from a freshly prepared sample. Spectra at t = 0 and 4 h present rather sharp signals, except for the solvent-exposed H N peaks ( Figure 6 upper panel), indicating the presence of species with small molecular weights and a disordered organization. This is not surprising as large oligomers and protofibrils cannot be observed by solution NMR due to fast relaxation, while disaggregated and/or small oligomers are NMR visible. 56,57 After 4 h, a slight decrease of signal intensity is observed ( Figure 6). This suggests that large aggregates are formed, although most of the peptide remains in the disaggregated and/or in small oligomer forms ( Figure 6).
A two-dimensional (2D) [ 1 H, 1 H] total correlation spectroscopy (TOCSY) spectrum reported in Figure S6 allows us to distinguish side chain protons of different residues of Aβ 25−35 . 1D [ 1 H] NMR spectra were also recorded for 1Pt dep alone at t = 0 and 4 h and are reported in Figure 7.
After 4 h, many changes occur in the spectrum: additional signals appear, and the peaks of the main compound decrease in intensity. New peaks are assigned to free ligands that are released from the metal coordination sphere. Indeed, previous 1 H NMR studies of the 1Pt analogue compound conducted in DMSO-d 6 indicated that both Dmphen and ethylene could be displaced by solvent molecules leading, over time, to squareplanar species. 48 In detail, the signal close to 0.0 ppm is due to Pt-CH 3 in a square-planar geometry, supporting the coex-istence of square-planar along with the bipyramidal trigonal geometry that, however, is still predominant after 4 h 48 ( Figure  7B).
1D [ 1 H] spectra were also acquired for Aβ 25 Figure 8A) and H α ( Figure 8B) protons but concern mainly with CH 2 (Figure 8C,D) and CH 3 ( Figure 8E) side chain protons, except for the serine residue that seems unaffected ( Figure 8B). The chemical shift changes indicate some   Figure  9A,G), while the presence of the metal complex at a 1:5 metal complex-to-peptide molar ratio determines the suppression of the fiber, favoring the formation of amorphous aggregates ( Figure 9D,J). SEM experiments corroborate these results: micrographs registered at different magnifications (reported in Figure 9) delight the presence of well-structured fibers with an average diameter of 3.3 ± 1.0 × 10 μm and a length of 4.7 ± 0.8 × 10 2 μm for Aβ 21−40 (Figure 9B,C) and a diameter of 24 ± 7 μm and a length of 8.1 ± 0.2 × 10 2 μm for Aβ 25−35 ( Figure  9H,I). The presence of 1Pt dep perturbs microstructure formation in this case as well: the microstructures appear immersed in the stub matrix, not a well defined and faintly visible event at high magnification values ( Figure 9E,F,K,L).
Fluorescence Assays. ThT fluorescence assays were performed at 25°C, employing a peptide concentration of 100 μM for Aβ 21−40 and 200 μM for Aβ 25−35 in 10 mM phosphate buffer at pH 7.4, using a ThT final concentration of 50 μM, at different ratios with Pt(II) complexes (stock solutions 1 mM in water for 1Pt dep and in 100% DMSO for both complexes). ThT experiments that have been carried out with 1Pt were acquired in solutions containing DMSO at 2% (v/ v). In this solvent, 1Pt is stable, as suggested by UV−vis absorption spectra collected as a function of time and reported in Figure S9. To   H] TOCSY spectrum was acquired with 58 scans, 128 free induction decays (FIDs) in t1, and 1024 data points in t2. Water suppression was achieved by presaturation. Spectra were processed and analyzed with TopSpin4.1.1 (Bruker, Italy). The 2D TOCSY spectrum was analyzed with NEASY 64 included in the Cara (computer-aided resonance assignment, http://www.nmr.ch/). Chemical shifts were referenced to the residual water peak at 4.75 ppm.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl Tetrazolium Bromide (MTT) Assay. Human SH-SY5Y neuroblastoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Paisley, U.K.) containing 10% heat-inactivated fetal bovine serum (FBS) (GIBCO), supplemented with 2 mM L-glutamine, 50 ng/mL streptomycin, and 50 units/mL penicillin and maintained in a humidified atmosphere (5% CO 2 at 37°C). Once at 70−80% of confluence, cells were harvested with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO). Aβ 21−40 and Aβ 25−35 alone or with 1Pt dep or 1Pt at a 1:5 molar ratio, respectively, were incubated in 50 mM sodium phosphate buffer, pH 7.2, under stirring, and samples were taken at three different times: 0, 2, and 24 h. Peptides were added to the cells in culture media in 96-well plates at 100 μM and then incubated for 24 h at 37°C. Cell viability was then assessed by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as previously described. 55,65 ■ CONCLUSIONS The present study well reflects our recent focus on the applicability of metal-based anticancer drugs in the field of neurodegeneration: by assuming different amyloidogenic peptides as model systems of neurodegenerative proteins, we assessed the ability of several metal complexes with different metal ions (such as Pt, Ru, and Au) and diverse geometries (square-planar and octahedral) to act as inhibitors of selfaggregation processes. [40][41][42]44 Herein, we have carried out several biophysical investigations to deepen, at the molecular level, the ability of two glycoconjugate Pt(II) bipyramidal complexes (Figure 1) to modulate amyloid aggregation. For its crucial involvement in AD, we have assumed as amyloid systems two fragments of the Aβ polypeptide: Aβ 21−40 and Aβ 25−35 . In the first assay, ThT fluorescence of the peptides in the absence and presence of metal compounds over time has been registered. Data indicated a clear suppression of the aggregation process of both the peptides, mostly by watersoluble 1Pt dep , with a greater effect on the inhibition of aggregation of Aβ 25−35 . ESI-MS and UV−vis absorption spectroscopy experiments outline that the inhibition of aggregation occurs through the formation of adducts between the Pt(II) bipyramidal complexes and Aβ peptides, through the insertion of peptides into the coordination sphere of the Pt center that implies the preferential release of the axial ligands, even if other exchanges can occur. ESI-MS analysis also indicated that in the case of the Aβ 25−35 /1Pt dep system, the metal complex can coordinate two peptide molecules at the same time, forming an adduct with 1:2 metal/peptide stoichiometry. This adduct cannot be formed in the case of Aβ 21−40 , probably because of its longer sequence. Conversely, Aβ 25−35 was the only sequence to provide an adduct, although of minimum intensity, with 1Pt. This complex has a lower intrinsic ability to form adducts with the peptides, probably because of its minor capacity, when compared to its deprotected analogue, to form hydrogen bonds that could be important in the early stage of the peptide/metal complex recognition process that precedes the formation of the coordinative bond. Because of limited ability of 1Pt to form adducts with the investigated peptides, we focused on 1Pt dep in further studies. From a conformational perspective, both CD and NMR experiments pointed out a deep mutual influence between the amyloidogenic peptide and 1Pt dep ; indeed, the presence of the metal complex stabilizes monomeric forms/ small aggregate forms of the peptide that do not evolve, during time, toward large aggregated species. In NMR assays, we observed that also the bipyramidal geometry of 1Pt dep is stabilized by the presence of Aβ 25−35 . Finally, microscopy investigations confirmed all spectroscopic data showing that 1Pt dep suppresses the formation of amyloid fibers for both Aβ sequences. Unfortunately, the Pt complexes investigated in this study are not able to rescue the cytotoxicity induced by amyloid peptides, as reported in Figure S10. Thus, these compounds cannot be directly translated as neurodrugs, but, instead, they can be assumed as valid templates to develop more specific drugs, preferentially able to cross the brain barrier.
In conclusion, this study represents an important example of how biophysical characterization of the adducts formed upon the reaction of metallodrugs with amyloid peptides can highlight on their mechanism of aggregation inhibition and is promising for the application of analogous glycoconjugate Pt(II) bipyramidal derivatives as novel therapeutics in neurodegenerative diseases.
Inorganic Chemistry pubs.acs.org/IC Article